148 European Journal of Environmental Sciences IN VITRO POLLEN GERMINATION OF ORCHIDS TRADITIONALLY USED TO PRODUCE SALEP

YASEMIN KEMEÇ1,*, KAAN HÜRKAN1, and CÜNEYT AKI2

1 Çanakkale Onsekiz Mart University, Institute of Natural and Applied Sciences, Department of Biology, 17100 Çanakkale, Turkey 2 Çanakkale Onsekiz Mart University, Faculty of Science and Arts, Department of Biology, 17100 Çanakkale, Turkey * Corresponding author: [email protected]

ABSTRACT

In Turkey the tubers of about 120 orchid species are widely collected for manufacturing the traditional drink salep. In this study, we focused on the in vitro germination of the pollen of the salep orchid species Ophrys mammosa, provincialis, subsp. morio, Orchis simia and tridentata and discussed the potential effects this might have on the conservation of these orchids by reducing the need to collect them in the field. Pollen was sown on different media; Knudson, Orchimax and the medium described by Malmgren, and then incubated at 24 ± 1 °C in darkness for 24 h. Germinated pollen was stained with Brilliant Blue and examined under a stereoscopic microscope. Results of Tukey and Dunnett T3 statistical tests indicated that in terms of percentage germination, the best germination was observed on O. mammosa by 55% and Orchimax was the most successful medium by 50.5%. For pollinaria germination, the best rate was observed on O. mammosa by 69%. The medium Malmgren was the best germinative by 61.3%. It is clearly seen that difference in germination rates among studied species are achieved using different media. The development of such a method of studied species in this research points to the fact that this is possible and should serve as encouragement for others to devise procedures for other species. These kinds of researches on propagation of orchids would be useful to reintroducing some of the rarer, endangered and endemic species in Turkey such previously succeed for and loeselii in Great Britain.

Keywords: pollinium, pollinarium, orchid, in vitro germination, salep, conservation

Introduction Around 120 orchid species, including the genera; Ace- ras, Anacamptis, Balia, Dactylorhiza, Himantoglossum, Orchids are cosmopolitan and occur in almost every Neotinea, Ophrys, Orchis and Serapias, are used to make habitat except in the Polar Regions (Edwards 2007). With salep in different places in Anatolia. Therefore, orchids more than 1000 genera and at least 25,000 species, the belonging to these genera are widely and intensively family is the largest and the most diverse collected from nature. In this study, we focused on the family of flowering (Harrap and Harrap 2009). orchids Ophrys mammosa (Desf.), Orchis provincialis While nearly 70% of orchids live on other plants as ep- (Balb.), Anacamptis morio (L.) R. M. Bateman, Pridgeon iphytes, they can also grow in soil (25%), and live on and M. W. Chase subsp. morio, Orchis simia (Lam.) and rocks and decaying plants (5%) (Arditti 1979; Renz and (Scop.) R. M. Bateman, Pridgeon Taubenheim 1984). In Turkey there are 170 terrestrial or- and M. W. Chase, which have been collected since an- chids belonging to 24 genera (Kreutz 2009). cient times in Anatolia and used to make salep (Özhatay In addition to their aesthetical and medicinal impor- et al. 1997; Baytop 1999). tance, orchids are ecological indicators (Joshi et al. 2009). Although orchids are protected worldwide from over- Moreover, orchids are used to manufacture a nutritious exploitation by the Convention on International Trade drink, called salep, which has been very popular for cen- in Endangered Species of Wild Fauna and Flora (CITES) turies in Anatolian and Arabian cultures and used as an (UNEP-WCMC 2013), they are still threatened by the additive in the production of “Maras” ice cream in Tur- illegal trade in orchids and the collecting of them from key (Sezik 1984; Baytop 1999; Kreutz 2009). nature to produce salep and “Maras” ice cream (Sezik et al. 2007; Kreutz 2009). It is estimated that 20–30 million orchids weighing 20 tons are collected from nature annu- Table 1 Tuber weights and tuber counts of orchids per 1000 kg (Sezik ally for producing salep (See Table 1). 1984). Because of the threat from logging, mining, urban- Commercial Average Number of tubers isation as a consequence of the increase in the human name tuber weight (g) per kg population, agricultural activities and the collecting of Muğla Salebi 0.23 4348 orchids from nature for the manufacture salep it is im- Kastamonu Salebi 0.50 2000 portant to develop new and advanced techniques for Silifke Salebi 0.35 2857 their sustainable usage (Kreutz 2009; Cribb 2011). This Antalya Salebi 0.21 4762 has resulted in improvements in the in vitro propagation of orchids in order to reduce the need to collect orchids Maraş Salebi 1.60 625 from nature and make it possible to conserve the most Van Salebi 1.00 1000 threatened species (Magrini et al. 2011). Thus, in this

Kemeç, Y., Hürkan, K., Aki, C.: In vitro pollen germination of orchids traditionally used to produce salep European Journal of Environmental Sciences, Vol. 5, No. 2, pp. 148–152 http://dx.doi.org/10.14712/23361964.2015.88 In vitro pollen germination of orchids traditionally used to produce salep 149

Table 2 List of the species studied, locations, dates of collection and numbers of pollinaria collected.

Collection location Collection of pollinaria Species (Çanakkale, Turkey) Date (2014) Number of pollinaria Anacamptis morio subsp. morio Kilitbahir April 15 210 Onsekiz Mart University Neotinea tridentata April 20 211 Terzioğlu Campus Ophrys mammosa Kilitbahir March 31 201 Orchis provincialis Denizgöründü village April 21 328 Orchis simia Denizgöründü village April 21 199

study, we developed a new method for the in vitro prop- M. W. Chase, growing wild in the centre of Çanakkale city agation using the pollen of orchids used in the manufac- and its surroundings (Turkey) (See Table 2 and Fig. 1). ture of salep. Plants were identified using “Flora of Turkey and the Although there are studies on the evolution (Brad- East Aegean Island” vol. 8 (Renz and Taubenheim 1984) shaw and Schemske 2003), ecology (Desrochers and and vol. 11 (Kreutz 2000), “Orkidelerimiz” (Sezik 1984) Rieseberg 1998), pollen ovule aging (Proctor 1998; Bel- and “Türkiye Orkideleri” (Kreutz 2009). lusci and Musacchio 2010), morphology and ultra-struc- ture of orchids (Feijo and Pais 1989; Pacini and Michael In vitro Germination of Pollen 2002) the germination pollen has not been previously Germination tests were started in March and April studied (Pritchard and Prendergast 1989; Aybeke 2002; 2014. The surfaces of pollinia were sterilized by placing Pacini and Michael 2002). them in a 0.5% solution of NaOC1 for 5 minutes after While many orchid genera have pollinia, which is an which they were rinsed three times in sterile distilled wa- adhesive mass of pollen, there are a few genera that pro- ter and between rinses they were broken into pieces in duce single pollen grains. Therefore, orchids differ great- a vortex mixer. ly in terms of whether they produce a mass of sticky pol- len or individual grains of pollen (Aybeke 2002). While the pollen of Pterostylis plumosa, Pterostylis concinna, Neottioids, Neuwiedia, Cypripedium acaule, Cypripedi- um calceolus, Apostasia wallachii consists of individual grains, that of Epipactis microphylla, Bletilla striata, Ne- ottia, Cleistes divaricata, Neottia nidus-avis, Epidendrum scutella, Epidendrum ibaguense, Loroglossum hircinum, Pleurothallis eumecocaulon and Calypso bulbosa con- sists of groups of four pollen grains (Pacini and Michael 2002). Using light-microscopy we observed that O. mam- mosa, O. provincialis, A. morio subsp. morio, O. simia and N. tridentata produce tetrad groups of pollens contained in pollinia. The aims of this study are to: (1) determine wheth- er it is possible to germinate the pollen of O. mammo- sa, O. provincialis, A. morio subsp. morio, O. simia and N. tridentata; (2) whether there differences in percentage germination between species and (3) the effects of KN, ORC and SV media on percentage germination.

Material and Methods

Collection of Study Materials Pollinia were collected from 5 species of orchids: Ophrys mammosa (Desf.), Orchis provincialis (Balb.), Fig. 1 Detailed images of flowers and pollinaria of N. tridentata and Anacamptis morio (L.) R. M. Bateman, Pridgeon and O. mammosa. Reproductive parts of Ophrys mammosa (A) and Neotinea M. W. Chase subsp. morio, Orchis simia (Lam.) and Neo­ tridentata (C). Pollinarium of Ophrys mammosa (B) and Neotinea tinea tridentata (Scop.) R. M. Bateman, Pridgeon and tridentata (D); p: pollinium, c: caudicle, v: viscidium.

European Journal of Environmental Sciences, Vol. 5, No. 2 150 Y. Kemeç, K. Hürkan, C. Aki

Table 3 Species studied, their codes used in subsequent tables and numbers of sown pollinia and pollinaria on individual media.

Number of sown pollinia Number of sown pollinaria Species Code KN ORC SV KN ORC SV Anacamptis morio subsp. morio A MOR 71 107 111 79 76 55 Neotinea tridentata N TRI 82 65 72 76 76 59 Ophrys mammosa O MAM 165 237 738 67 62 71 Orchis provincialis O PRO 119 84 121 113 123 92 Orchis simia O SIM 72 75 81 50 67 82

Two different commercial media (Orchimax Orchid were significant differences between N TRI, O MAM and Medium “ORC”, O0257 and Knudson C Orchid Medi- O SIM (Table 4). Overall germination rates of individ- um “KN”, K0215, Duchefa Biochemie BV, Haarlem, the ual media for pollinia are shown in Table 5. There were Netherlands) and one special medium (SV) (Malmgren no significant differences in germination rates of pollinia 2006) were used. KN and ORC media were supplement- among media (Table 5). Overall pollinia germination ed with 20 g/l sucrose, 1 g/l activated charcoal and 6 g/l rates for individual species are shown in Table 6. The dif- agar. SV was prepared with; 90 mg/l Ca3(PO4)2, 90 mg/l ferences in germination rates between species were not KH2PO4, 90 mg/l MgSO4, 20 g/l sucrose, 1 g/l activated statistically significant (Table 6). charcoal, 6 g/l agar and 30 ml/l pineapple juice. The pH of We observed germination within the inner sides of the medium was adjusted to 5.75 ± 0.1 before autoclaving pollinaria where pollinia were connected. Pollen tubes at 121 °C and 101 kPa for 20 min. Approximately 50–200 increasingly elongated from inside to outside (Fig. 3). pollinia (Table 3) were sown under axenic conditions in Statistical analyses confirmed significant differences of each Petri dish (10 cm in diam.), containing 25 ml of ei- species-media composition and species-media interac- ther KN, ORC or SV medium. There were five replicates tion on germination rates (p < 0.05). for each accession of pollinia. The Petri dishes were sealed The best germination rates of pollinaria for O PRO and with stretch film, wrapped in aluminium foil to exclude O SIM were on the medium KN, for N TRI and O MAM light and incubated at 24 ± 1 °C in darkness for 24 h. on the medium SV, for A MOR on the medium ORC The pollen was stained with Brilliant Blue R (Sig- (Table 7). There were statistically significant differenc- ma-Aldrich B7920) and the number that had germinat- es between individual media for O MAM, A MOR and ed counted under a stereoscopic microscope (Olympus N TRI, but there was none between O PRO and O SIM SZ-51). (Table 7). Overall germination success rates of pollinar- ia for individual media are shown in Table 8; there was Statistical Analyses no significant difference between the media. The overall Results were analyzed using one-way ANOVA (IBM germination rates of pollinaria for individual species are SPSS Statistics ver. 22), followed by Tukey ’ s HSD and shown in Table 9. There were no statistically significant Dunnet T3 tests to determine whether the results differed differences between these. significantly for the different orchids and media.

Results

We observed that germination occurred solely be- tween connecting surfaces of pollinia to pollinaria and pollen tubes were elongated such fringes from pollen grains while there was no germination observed on outer surfaces of pollinia (Fig. 2). We also investigated Species + Media interaction effects on germination rate and performed statistical analyses. Statistical analyses showed that Species + Media interaction was significant at p < 0.05. The best germination rates of pollinia for A MOR and N TRI were on the medium KN, for O MAM and O PRO on the medium ORC and for O SIM on the medium SV Fig. 2 Pollen tube (p) development on pollinia of Ophrys mammosa (A), (Table 4). The germination rates, however, did not sta- Orchis simia (B), Neotinea tridentata (C) and Orchis provincialis (D). Scale tistically differ between A MOR and O PRO, but there bars: 1 mm.

European Journal of Environmental Sciences, Vol. 5, No. 2 In vitro pollen germination of orchids traditionally used to produce salep 151

Table 4 Germination rates of pollinia (mean ± SD) of individual species Table 8 Overall pollinaria germination rates (mean ± SD) on each on each medium. medium.

KN ORC SV p value F2,12 mean ± SD p value F2,12 A MOR 39.6 ± 5.5 32.7 ± 5.3 36.9 ± 11.6 0.413 0.952 KN 57.2 ± 13.0 N TRI 51.2 ± 4.3 40.9 ± 6.3 44.4 ± 6.8 0.049 3.903 ORC 56.8 ± 6.1 0.661 0.428 O MAM 54.4 ± 3.6 65.6 ± 8.7 52.3 ± 3.0 0.007 7.760 SV 62.7 ± 13.3 O PRO 42.0 ± 9.8 46.6 ± 12.1 37.1 ± 15.6 0.508 0.716 O SIM 28.2 ± 9.3 46.8 ± 7.2 50.6 ± 8.4 0.002 10.367 Table 9 Overall pollinaria germination rates (mean ± SD) for individual species.

mean ± SD p value F4.10 Table 5 Overall germination rates of pollinia (mean ± SD) on each A MOR 59.3 ± 10.4 medium. N TRI 46.2 ± 4.9

mean ± SD p value F2,12 O MAM 68.3 ± 14.9 0.588 0.737 KN 43.4 ± 10.9 O PRO 54.5 ± 5.6 ORC 46.6 ± 11.0 0.885 0.124 O SIM 56.6 ± 5.4 SV 44.4 ± 9.5

Discussion

Table 6 Overall pollinia germination rates (mean ± SD) for individual Although in vitro symbiotic and asymbiotic seed ger- species. mination has become a favoured and useful technique

mean ± SD p value F4.10 for orchid propagation and use in reintroduction A MOR 36.1 ± 3.4 to nature, in vitro pollinium or pollinarium germination N TRI 46.2 ± 4.9 has not been studied widely in terrestrial orchids. This is O MAM 56.7 ± 5.5 0.060 3.233 especially important for species used for commercial sa- lep production. Therefore, here we provide the first suc- O PRO 42.0 ± 6.1 cessful in vitro pollinia and pollinaria germination and O SIM 42.5 ± 4.9 comparison report of salep orchids Ophrys mammosa, Orchis provincialis, Orchis simia and Neotinea tridentata. All plants, which have pollinium instead of pollen grains, need water for hydration of complex pollen clus- ters (Marginson et al. 1985). During pollinium devel- opment of Epidendrum ibaguense, plasmodesmata and thin cytoplasmic channel formations enclose tetrads, and consequently proximal sides of each tetrad have thinner walls (Yeung 1987). This structural differentiation helps water to be absorbed easier, which explains germination success of inner parts of pollinaria. As observed recently (Yeung 1987; Feijo and Pais 1989; Aybeke 2002), for both Fig. 3 Pollen tube (p) development and pollinium (l) view on pollinarium pollinia and pollinaria, only inner surfaces where pol- of Ophrys mammosa (A) and Anacamptis morio subsp. morio (B). Scale linia were attached to pollinaria, germinated and pollen bars: 1 mm. tubes were developed. It can be explained by hydration advantages of pollen tetrads at broken parts of pollini- um during the breaking processes of pollinaria, and by thinner walls of proximal sides of tetrads. Therefore, be- Table 7 Germination rates of pollinaria (mean ± SD) for each medium fore sowing process, breaking pollinaria into pollinia and and species. even smaller pieces can increase the success of germina-

KN ORC SV p value F2,12 tion significantly. These results are parallel with otherin A MOR 38.4 ± 8.2 66.4 ± 12.9 59.9 ± 17.6 0.016 5.973 vivo results regarding germination differences between N TRI 52.1 ± 6.0 54.6 ± 8.2 69.2 ± 8.6 0.009 7.135 inner and outer walls of tetrads (Swamy 1947; Podduban- O MAM 51.6 ± 19.8 49.9 ± 6.9 81.7 ± 14.7 0.015 6.041 ya-Arnoldi 1976). Our comparison of pollinia and polli- naria germination supports those results. O PRO 57.5 ± 13.7 52.5 ± 15.9 48.4 ± 4.7 0.524 0.683 It is known that sugar level of germination medium O SIM 61.9 ± 11.2 56.7 ± 13.1 51.2 ± 5.9 0.309 1.297 is very important for germination as well. While Pfundt

European Journal of Environmental Sciences, Vol. 5, No. 2 152 Y. Kemeç, K. Hürkan, C. Aki

(1909) recommends sucrose level by 5–20% (w/v), an- Feijo JA, Pais MSS (1989) Cytomixis in meiosis during the micro- other researcher found orchid pollen germinates best on sporogenesis of ophrys lutea: An ultrastructural study. Caryo- the medium with a range of 3–10% (w/v) sucrose level logia 42: 37–48, doi:10.1080/00087114.1989.10796951. (Miwa 1937). Relatively recently, 1–10% (w/v) sugar level Harrap A, Harrap S (2009) Orchids of Britain and Ireland. 2nd ed. A and C Black Publ. Ltd., London. was suggested (Pritchard and Prendergast 1989). Con- Joshi GC, Tewari LM, Lohani N, Upreti K, Jalal JS, Tewari G (2009) sidering this and according to our pilot experiments we Diversity of orchids on Uttarakhand and their conservation decided 2% (w/v) sucrose level as the optimum for each strategy with special reference to their medicinal importance. germinating medium in our study. Report and Opinion 1: 47–52. We show that Svante medium can be used as a basic Kreutz KCAJ (2000) Orchidaceae. In: Davis PH (ed) The flora germinating medium for orchids Ophrys mammosa, Or- of Turkey and the east Aegean islands. University Pres, Edin- chis provincialis, Anacamptis morio subsp. morio, Orchis burgh, pp. 275–375. simia and Neotinea tridentata. Future studies can devel- Kreutz KCAJ (2009) Türkiye orkideleri. Rota Yayınları, Istanbul. Magrini S, Carli AD, Onofri S, Scoppola A, De Carli A (2011) op germination medium with different combinations of A comparative study of the seed germination capabilities of supplements and investigating medium success on more Anacamptis palustris (Orchidaceae), a threatened terrestrial orchid species. Future studies must be aimed to ensure orchid, and other more common anacamptis. Eur J Environ Sci further development of the plants until adulthood for 1: 71–79. further reintroduction to nature and manufacturing sa- Malmgren S (2006) Orchid propagation. www.lidaforsgarden.com lep from their tubers. /Orchids/engelsk.htm. Accessed 12 September 2014. Marginson R, Sedgley M, Knox RB (1985) Physiology of post-pol- lination exudate production in acacia. J Exp Bot 36: 1660–1668. Miwa A (1937) Test of the germinating power of orchid pollen. Acknowledgements Orchid Rev 45: 9. Özhatay N, Koyuncu M, Altay S, Byfield A (1997) Türkiye ’ nin The authors wish to thank Cathy Seither for linguistic doğal tıbbi bitkilerinin ticareti hakkında bir çalışma. Doğal improvement of this manuscript and Svante Malmgren Hayatı Koruma Derneği, Istanbul. for sharing his own-developed medium receipt. Pacini E, Michael H (2002) Types of pollen dispersal units in or- chids, and their consequences for germination and fertilization. Ann Bot 89: 653–664. doi:10.1093/aob/mcf138. Pfundt M (1909) Der Einfluss der Luftfeuchtigkeit auf die Lebens- REFERENCES dauer des Blütenstaubes. Borntraeger, Leipzig. Poddubanya-Arnoldi VA (1976) Cytoembryology of angiosperms. Nauka Publishing, Moscow. Arditti J (1979) Aspects of the physiology of orchids. 1st ed. Ad- Pritchard HW, Prendergast FG (1989) Factors influencing the ger- vances in Botanical Research, New York. mination and storage characteristics of orchid pollen. Modern Aybeke M (2002) In vitro germination experiments on granular methods in orchid conservation. Cambridge University Press, pollens and polliniums in orchids. Journal of the Institute of Cambridge. Science and Technology Gazi Uni 15: 71–79. Proctor HC (1998) Effect of pollen age on fruit set, fruit weight, Baytop T (1999) Türkiye ’ de bitkilerle tedavi: Geçmişte ve bugün. and seed set in three orchid species. Can J Bot 76: 420–427. Nobel Tıp Kitapevleri, Istanbul. Renz J, Taubenheim G (1984) Orchidaceae. In: Davis PH (ed) The Bellusci F, Musacchio A (2010) Differences in pollen viability in flora of Turkey and the east Aegean islands. 8th ed. Edinburgh relation to different deceptive pollination strategies in Med- University Press, Edinburgh, pp. 450–552. iterranean orchids. Ann Bot 106: 769–774, doi:10.1093/aob Sezik E (1984) Orkidelerimiz Türkiye ’ nin orkideleri. Sandoz /mcq164. Kültür Yayınları, Istanbul. Bradshaw HD, Schemske DW (2003) Allele substitution at a flower Sezik E, İşler S, Orhan Ç, Deniz Gİ, Güler N, Aybeke M, Üstün colour locus produces a pollinator shift in monkeyflowers. Na- O (2007) Salep ve orkidelerin tahribi. The Scientific And Tech- ture 426: 176–178, doi:10.1038/nature02106. nological Research Council Of Turkey, Ankara. Cribb P (2011) Orchids in a changing climate. Lankesteriana 11: Swamy BGL (1947) On the life history of Vanilla planifolia (Orchi- 233–238. daceae). Bot Gaz: 449–455. Desrochers AEM, Rieseberg LH (1998) Effects in wild species of UNEP-WCMC (2013) The checklist of cites species website. http:// (Asteraceae). Amer J Bot 85: 770–775. www.cites.org/. Accessed 20 October 2014. Edwards N (2007) Flowers see how plants grow. PowerKids Press, Yeung EC (1987) Mechanisms of pollen aggregation into pollinia Mishawaka, IN, USA. in Epidendrum ibaguense (Orchidaceae). Grana 26: 47–52.

European Journal of Environmental Sciences, Vol. 5, No. 2